1. Prague 2008 1 Plasma vs. tissue concentration to predict antibiotic efficacy PL Toutain UMR 181 Physiopathologie et Toxicologie Experimentales INRA/ENVT ECOLE NATIONALE VETERINAIRE T O U L O U S E Fourth International conference on AAVM Prague, Czech republic 24-28, 2008

2. Prague 2008 2 In veterinary medicine, there are many publications on tissular concentrations to promote the idea that some antibiotics having a high tissular concentration accumulate in biophase (quinolones, macrolides) and are more efficacious as suggested by their low or undetectable plasma concentrations The inadequate tissue penetration hypothesis

3. Prague 2008 3 Two false assumptions 1.tissue is homogenous 2.bacteria are evenly distributed through tissue  spurious interpretation of all important tissue/serum ratios in predicting the antibacterial effect of AB The inadequate tissue penetration hypothesis: Schentag 1990 Schentag, 1990

4. Prague 2008 4 Statements such as ‘concentrations in tissue x h after dosing are much higher than the MICs for common pathogens that cause disease’ are meaningless Mouton & al JAC 2007

5. Prague 2008 5 Objectives of the presentation: To address some basic questions 1.Where are located the bugs ? Extracellular vs. intracellular 2.Where is the biophase? Interstitial space fluid vs. intracellular cytosol vs. intracellular organelles 3.What is a tissue and what is a tissue concentration 4.How to assess the biophase antibiotic concentration Total tissular concentration vs. ISF concentration. 5.The issue of lung penetration 1.Epithelial lining fluid (ELF):???? 2.The hypothesis of targeted delivery of the active drug at the infection site by phagocytes 6.Plasma as the best surrogate of biophase concentration for PK/PD interpretation

6. Prague 2008 6 Q1: Where are located the pathogens

7. Prague 2008 7 Where are located the pathogens ISF Most bacteria of clinical interest S. pneumoniae E. coli Klebsiella Mannhemia ; pasteurella Actinobacillius pleuropneumoniae Mycoplasma hyopneumoniae Bordetala bronchiseptia Cell ( most often in phagocytic cell) mycoplasma (some) Chlamydiae Brucella Cryptosporidiosis Listeria monocytogene Salmonella Mycobacteria Rhodococcus equi

8. Prague 2008 8 Q2: Where is the biophase

9. Prague 2008 9 The interstitial space fluid is the biophase 1.Most bacterial infections are located in the extracellular compartment. 2.Except few cases, In acute infections in non- specialized tissues, where there is no abscess formation, interstitial space fluid (ISF) must be considered as the actual target space for anti- infective agents 3.ISF concentrations are of primary interest Muller et al. AAC, 2004, 48: 1441-1453

10. Prague 2008 10 Q3: what is a tissue & what is a tissular concentration

11. Prague 2008 11 Historical definition of a tissue drug concentration In the past, it was used to characterize the total concentration in a homogenized biopsy sample –For vet medicine: a by-product of regulatory residue studies It was assumed that: – tissue is homogenous –that antibiotics is evenly repartited in tissue –That bacteria are evenly repartited in tissue –Each of these assumptions is false and can be very misleading

12. Prague 2008 12 Why a total drug tissue concentrations may be misleading? 1.Drug distributed mainly extracellularly β-lactams and aminoglycosides, grinding up the tissue means dilution of the drug by mixing intracellular and extracellular fluids, resulting in underestimation of its concentrations at the site of infection. 2.Drug accumulated by cells fluoroquinolones or macrolides assay of total tissue levels will lead to gross overestimation of the extracellular concentration. The opposite is true for intracellular infections.

13. Prague 2008 13 Methods for studies of target site drug distribution in antimicrobial chemotherapy

14. Prague 2008 14 “Tissue concentrations” Total tissue –homogenates –biopsies Extracellular fluids –implanted cages –implanted threads –wound fluid –blister fluid –ISF (Microdialysis, Ultrafiltration)

15. Prague 2008 15 The tissue cage model for in vivo and ex vivo investigations

16. Prague 2008 16 The tissue cage model Perforated hollow devices Subcutaneous implantation development of a highly vascularized tissue fill up with a fluid with half protein content of serum (delay 8 weeks) C.R. Clarke. J. Vet. Pharmacol. Ther. 1989, 12: 349-368

17. Prague 2008 17 The tissue cage model : PK limitations A foreign body –Not a physiological space –Clinical counterparts? Ascitic fluid, effusions ( pericardial, pleural…) Interpretation may be difficult because PK determined by: –diffusion capacity across the TC – TC size and geometry surface area/volume ratio is the major determinant of peak and trough drug level

18. Prague 2008 18 The tissue cage model Drug administration Slow equilibration inoculation Time (C) Time (C) T1/2 varies with the surface area / volume ratio of the tissue cage Penicillin 5 to 20 h Danofloxacin 3 to 30 h Greko, 2003, PhD Thesis

19. Prague 2008 19 Microdialysis & ultrafiltration Techniques

20. Prague 2008 20 What is microdialysis (MD)? Microdialysis, a tool to monitors free antibiotic concentrations in the fluid which directly surrounds the infective agent

21. Prague 2008 21 Microdialysis: The Principle The MD Probe mimics a "blood capillary". There is an exchange of substances via extracellular fluid Diffusion of drugs is across a semipermeable membrane at the tip of an MD probe implanted into the ISF of the tissue of interest.

22. Prague 2008 22 the implanted MD probe is perfused with the perfusate, ie, a physiologic liquid at a very slow rate. Substances present in the interstitial space fluid of the investigated site can diffuse into the perfusate through a semipermeable membrane at the tip of the MD probe and appear in the dialysate. Afterward the concentration in the dialysate is chemically analyzed and the true concentration in the interstitial space fluid can be calculated. Microdialysis: The Principle Antibiotic

23. Prague 2008 23 Microdialysis materials CMA60 Microdialysis 1.Introducer with CMA 60 Microdialysis Catheter 2.Outlet tube 3.Vial holder 4.Microvial 5.Inlet tube 6.Luer lock connection 7.Puncture needle.

24. Prague 2008 24 Microdialysis : Limits MD need to be calibrated Retrodialysis method –Assumption: the diffusion process is quantitatively equal in both directions through the semipermeable membrane. –The study drugs are added to the perfusion medium and the rate of disappearance through the membrane equals in vivo recovery. – The in vivo percent recovery is calculated (CV of about 10-20%)

25. Prague 2008 25 A small experimental error in recovery estimate results in a relatively larger error in drug concentration estimates which is probably responsible for the greater interanimal variability observed in lung tissue than in the other media Marchand & al AAC June 2005 MD need to be calibrated:

26. Prague 2008 26 Ultrafiltration Excessive (in vivo) calibration procedures are required for accurate monitoring Unlike MD, UF- sample concentrations are independent on probe diffusion characteristics

27. Prague 2008 27 Microdialysis vs. Ultrafiltration Ultrafiltration Vacuum Microdialysis : a fluid is pumped through a membrane; The driving force is a pressure differential (a vacuum) applied across the semipermeable membrane The analyte cross the membrane by diffusion The driving force is a concentration gradient

28. Prague 2008 28 Marbofloxacin : plasma vs.ISF In vivo filtration Bidgood & Papich JVPT 2005 28 329 Microdialysis Not suitable for long term in vivo studies Ultrafiltration Suitable for long term sampling (in larger animals, the UF permits complete freedom of movement by using vacutainer collection method)

29. Prague 2008 29 What we learnt with animal and human microdialysis studies

30. Prague 2008 30 Plasma (total, free) concentration vs interstitial concentration (muscle, adipose tissue) (Moxifloxacin) Muller AAC, 1999 Time (h) Total (plasma, muscle) free (plasma) interstitial muscle interstitial adipose tissue 261012 30 40 20 100 1000 Concentration (ng/mL)

31. Prague 2008 31 Plasma (total, free) concentration vs muscle (free) concentration Total (plasma) free (muscle) free (plasma) Liu J.A.C. 2002 cefpodoxine cefixime

32. Prague 2008 32 What we learnt with animal and human MD studies MD studies showed that: 1. the concentrations in ISF of selected antibiotics correspond to unbound concentrations in plasma 2.They are generally much lower than total concentrations reported from whole-tissue biopsy specimens especially for macrolides and quinolones

33. Prague 2008 33 What we learnt with MD studies: Inflammation

34. Prague 2008 34 Tissue concentrations of levofloxacin in inflamed and healthy subcutaneous adipose tissue Methods: Free Concentrations measured in six patients by microdialysis after administration of a single intravenous dose of 500 mg. Results:The penetration of levofloxacin into tissue appears to be unaffected by local inflammation. Same results obtained with others quinolones Hypothesis: Accumulation of fibrin and other proteins, oedema, changed pH and altered capillary permeability may result in local penetration barriers for drugs Bellmann & al Br J Clin Pharmacol 2004 57 Inflammation No inflammation

35. Prague 2008 35 What we learnt with MD studies: Inflammation Acute inflammatory events seem to have little influence on tissue penetration. “These observations are in clear contrast to reports on the increase in the target site availability of antibiotics by macrophage drug uptake and the preferential release of antibiotics at the target site a concept which is also used as a marketing strategy by the drug industry” Muller & al AAC May 2004

36. Prague 2008 36 In acute infections in non- specialized tissues, where there is no abscess formation, free serum levels of antibiotics are good predictors of free levels in tissue fluid

37. Prague 2008 37 The issue of lung penetration

38. Prague 2008 38 Animal and human studies MD: The issue of lung penetration Lung MD require maintenance under anesthesia, thoracotomy (patient undergoing lung surgery).. Does the unbound concentrations in muscle that are relatively accessible constitute reasonable predictors of the unbound concentrations in lung tissue (and other tissues)?

39. Prague 2008 39 Free muscle concentrations of cepodoxime were similar to free lung concentration and therefore provided a surrogate measure of cefpodoxime concentraion at the pulmonary target site Liu et al., JAC, 2002 50 Suppl: 19-22. Cefpodoxime at steady state: plasma vs. ISF (muscle & Lung) Plasma Free plasma Muscle Lung

40. Prague 2008 40 The issue of lung penetration: Imipem The major finding of this study was the observation of virtually superimposed free IPM concentration-versus-time profiles in the three media investigated, This result not only is in agreement with theory but also is consistent with most of the data in the literature. imipenem distribution in muscle and lung interstitial fluids Marchand & al AAC June 2005

41. Prague 2008 41 The issue of lung penetration

42. Prague 2008 42 Lung infections Uncertainty of the relevant actual location of proliferating bacteria –Alveoli, pulmonary interstitium, bronchioles, blood?? What is the biophase?? –Epithelium lining fluid (ELF) –Lung IF, alveolar macrophages, tisue biopsies, blood, bronchial secretion, sputum?? ELF seems the most relevant specimen but potential sources of error: dilution, release of AB from alveolar macrophage in the sample

43. Prague 2008 43

44. Prague 2008 44 The blood-alveolar barrier The alveolar epithelial cells would not be expected to permit passive diffusion of antibiotics between cells, the cells being linked by tight junctions Fenestrated pulmonary capillary bed expected to permit passive diffusion of antibiotics with a molecular weight 1,000 Epithelial lining fluid ELF (protein:<10%)

45. Prague 2008 45 Drug passage in the lung Drug passage through the alveolar epithelial cells will depend on the lipophilicity and diffusibility of the antibiotics, similar to the drug entry into the central nervous system. Kiem & Schentag AAC 2008 Jan 24-36 ELF

46. Prague 2008 46 ELF concentration: possible biais Measurement problems may confound the interpretation of the ELF concentrations of antibiotics. Cells, especially AM cells (that constitute 3.8 to 10.0% of ELF volume) are included in the composition of ELF The cells may be lysed during the measurement of antibiotic concentration in BAL-derived fluids. Kiem & Schentag AAC 2008 Jan 24-36 ELF

47. Prague 2008 47 BETA-LACTAMS Measured ELF concentrations of the beta-lactams are well below serum concentrations, and their respective concentrations in AM cells were negligible The low measured ELF concentrations of betalactams in comparison to their corresponding serum levels could be the result of low capacity of their unbound free fractions for penetration through the alveolar epithelial cell barriers. Kiem & Schentag AAC 2008 Jan 24-36 ELF

48. Prague 2008 48 MACROLIDES AND KETOLIDES Measured ELF concentrations of macrolides and ketolides and their derived AUCs were consistently higher than serum levels by as much as 10-fold the high ratios of ELF concentration to serum concentration for macrolides and ketolides could not be explained solely on the basis of good penetration across the alveolar epithelium. The high concentrations of macrolides and ketolides in ELF might be explained by the possible contamination of intracellular antibiotics occurring during the process of BAL. Kiem & Schentag AAC 2008 Jan 24-36 ELF

49. Prague 2008 49 FLUOROQUINOLONES Fluoroquinolones achieved higher ELF levels than their free serum concentrations Kiem & Schentag AAC 2008 Jan 24-36

50. Prague 2008 50 The high ELF concentrations of some antibiotics, which were measured by the BAL technique, might be explained by possible contamination from high achieved intracellular concentrations and subsequent lysis of these cells during the measurement of ELF content. This effect is similar to the problem of measuring tissue content using homogenization Kiem & Schentag’ Conclusions (1)

51. Prague 2008 51 Fundamentally, ELF may not represent the lung site where antibiotics act against infection. In view of the technical and interpretive problems with conventional ELF and especially BAL, the lung microdialysis experiments may offer an overall better correlation with microbiological outcomes. We continue to express PK/PD parameters using serum concentration of total drug because these values do correlate with microbiological outcomes in patients. Kiem & Schentag’ Conclusions (2)

52. Prague 2008 52 The site of infection: Intracellular pathogens

53. Prague 2008 53 Intracellular location of bacteria Phagosome Lysosome Chlamydiae Listeria No fusion with lysosome Phagolysosome S.aureaus Brucella Salmonella Coxiella burneti pH=5.0 3 4 2 1 Fusion pH=7.4 B B B B B B B B Cytosol

54. Prague 2008 54 Intracellular location of antibiotics Phagolysosome volume 1 to 5% of cell volume pH=5.0 Macrolides (x10-50) Aminoglycosides (x2-4) Cytosol pH=7.2 Fluoroquinolones(x2-8) beta-lactams (x0.2-0.6) Rifampicin (x2) Aminoglycosides (slow) Ion trapping for weak base with high pKa value

55. Prague 2008 55 What are the antibiotic intracellular expressions of activity Phagolysosome Macrolides Aminoglycosides Cytosol pH=7.2 Fluoroquinolones beta-lactams Rifampicin Aminoglycosides Good Low or nul

56. Prague 2008 56 The hypothesis of targeted delivery of active drug at the active site by the phagocytes

57. Prague 2008 57 Drug delivery by the phagocytes Transport by "non professional" phagocytes (e.g. azithromycin by fibroblast) Fibroblast acts as a reservoir for drug and macrophages

58. Prague 2008 58 Neutrophils as antibiotic delivery system The ability of neutrophils to migrate preferentially to sites of infection makes them attractive as a delivery mechanism for antibiotics; theoretically, with the proper cellular PK, an antibiotic could be taken up by neutrophils, which would then transport the drug and later release it. This could provide a mechanism for achieving higher levels of antibiotic in tissues i.e. directly at the nidus of infection. Scorneaux & Shryock tilmicosine in pigs; JVPT 1998, 21: 257-258 & tilmicosine in cattle in: J. Dairy Sci. 1999, 82: 1202-1212)

59. Prague 2008 59 Macrolides: how to explain efficacy of low plasma concentrations? M M 1.PK hypothesis 2-PD hypothesis M PMN Nucleus High local M concentration in the vicinity of the bug Sink or reservoir???

60. Prague 2008 60 PK model for exposure to macrolides: The limits M+M+ M M M MMM M M 2.Slow or very slow efflux (e.g. 20% within 3 h for azithromycin) more rapid (90% within 1 h) for erythromycin) Transit time in a (normal) lung capillary: 26 sec No In vivo data to support the hypothesis Mass balance considerations (Tylosine in piglet 10 Kg BW, dose: 10 mg/kg) Total pool of PMN=2 mL/L of blood Peak Plasma concentration: 1 µg/mL Accumulation ratio: 50 Total amount of drug located in PMN :100µg in toto =1/1000 of the dose Sink rather than reservoir

61. Prague 2008 61 Macrolides: how to explain efficacy of low plasma concentrations? M M 1.PK hypothesis 2-PD hypothesis M PMN Nucleus MIC in vivo < MIC in vitro Cytokine modulation, ERK signaling High local M concentration in the vicinity of the bug M Sink or reservoir???

62. Prague 2008 62 Conclusions: 1.In acute infections in non-specialized tissues, where there is no abscess formation, free plasma levels of antibiotics are good predictors of free levels in interstitial fluid 2.PK/PD indices predictive of antibiotic efficacy should be based on free plasma concentration 3.People who truly understand tissue concentration work in corporate marketing departments (Apley, 1999)